|Year : 2014 | Volume
| Issue : 2 | Page : 88-93
Characteristic cytoplasmic morphology of oocytes in endometriosis patients and its effect on the outcome of assisted reproduction treatments cycles
Ayse Kendirci Ceviren1, Neval Tanriverdi Ozcelik1, Aysenur Urfan1, Levent Donmez2, Mete Isikoglu1
1 Gelecek The Center for Human Reproduction, Antalya, Turkey
2 Department of Preventive Medicine and Public Health, Akdeniz University, Medical School, Antalya, Turkey
|Date of Web Publication||4-Sep-2014|
Gelecek The Center for Human Reproduction, Antalya
Source of Support: None, Conflict of Interest: None
Introduction: Endometriosis is a clinical disease that is associated with poor outcomes in in vitro fertilization programs with a decrease in oocyte retrieval, oocyte quality, implantation and pregnancy rates.Based on the observations that the majority of the oocytes obtained in patients diagnosed with endometriosis have some characteristics, we conducted a retrospective study to reveal a more definite picture and to establish a simple way of prediction of the outcome. Materials and Methods: The patients were triaged in two groups based on the diagnosis of endometriosis. After denudation, the oocytes were examined by inverted microscope with ×200 magnification just before intracytoplasmic sperm injection. Oocytes with a centrally dark and granular cytoplasm were diagnosed to have degenerative signs. Results: Data of 792 patients were reviewed retrospectively. The percentage of oocytes with degenerative signs is significantly higher in endometriosis (27.4%) group compared with control group (7.9%) ( P < 0.001). Although, there is a tendency to lower pregnancy rate in endometriosis group (38.7%) the difference is not statistically significant (50.4%) ( P = 0.077). Conclusion: The oocytes have a characteristic appearance of dark and granular ooplasm in endometriosis cases. This finding can be used as a clue in cases in which the presumptive diagnosis has not verified by laparoscopy. The typical morphology also has an adverse effect on clinical outcome.
Keywords: Assisted reproduction, endometriosis, oocyte quality
|How to cite this article:|
Ceviren AK, Ozcelik NT, Urfan A, Donmez L, Isikoglu M. Characteristic cytoplasmic morphology of oocytes in endometriosis patients and its effect on the outcome of assisted reproduction treatments cycles. IVF Lite 2014;1:88-93
|How to cite this URL:|
Ceviren AK, Ozcelik NT, Urfan A, Donmez L, Isikoglu M. Characteristic cytoplasmic morphology of oocytes in endometriosis patients and its effect on the outcome of assisted reproduction treatments cycles. IVF Lite [serial online] 2014 [cited 2022 Jan 18];1:88-93. Available from: http://www.ivflite.org/text.asp?2014/1/2/88/140123
| Introduction|| |
From the beginning of the introduction of assisted reproduction treatments (ART), efforts focused on the prediction of the treatment outcome as well as the improvement of the success rates. In order to predict the in vitro fertilization (IVF) outcome, numerous studies investigated the morphological assessment of the pre-implantation stage embryos, the hormonal environment and the various biomarkers in follicular fluid, serum or peritoneal fluid.  Several studies also focused on the meiotic and chromosomal patterns in pre-implantation phase. Among several options, routine morphological assessment of pre-implantation stage embryos through inverted microscope is a simple, cost effective and most widely used method and performed routinely in the majority of the IVF clinics so far. For this aim, certain criteria are applied at various stages of development. Recently, Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology revealed a consensus report, which was intended to be referenced as a global consensus to allow standardized reporting of the minimum data set required for the accurate description of embryo development.  Based on the morphological assessment of the embryo, ~20-25% of embryos transferred on day 2 or day 3 produce a baby, and the implantation rate (IR) reaches ~30-40% with transfer at the blastocyst stage. 
Assessment of oocyte morphology is also an important task since deviations from normal may affect embryo development and its potential for implantation and healthy pregnancy/delivery. , Only ~5% of fresh oocytes produce a baby. This low rate decreases to 1% in older mothers and reaches its peak, 7%, in oocyte donation programs.  Since the quality of the oocyte has a crucial role not only in fertilization, but also in subsequent development of the embryo,  studies also investigated the evaluation of oocyte characteristics including the cytoplasm, the perivitelline space and the zona pellucida in intracytoplasmic sperm injection (ICSI) cycles.
Endometriosis is a clinical disease that is associated with poor outcomes in IVF programs with a decrease in oocyte retrieval, oocyte quality, IR and pregnancy rates.  The existing studies in the literature focus on the effect of endometriosis on the further embryo development and the ART outcome parameters. Particularly ovarian dysfunction, immunologic defects and altered characteristics of the peritoneal environment including certain cytokines and mediators are frequently studied.
Based on the observations that the majority of the oocytes obtained in endometriosis patients have certain morphological characteristics, we conducted a retrospective study to demonstrate the relationship of some special features of the oocytes with endometriosis and to reveal the effect of these morphological abnormalities on ICSI outcome.
| Materials and Methods|| |
The study was conducted at a private fertility clinic between February 2009 and December 2011.
Laboratory archives of all ICSI treatments were retrospectively reviewed. Each patient in the study contributed a single cycle.
All treatment cycles were monitored by the same clinician and controlled ovarian hyperstimulation protocols were tailored in accordance with the findings of each patient. They included the standard long protocol with a daily gonadotropin-releasing hormone agonist (GnRH) agonist for 351 patients, microdose flare up protocol with a daily GnRH agonist for 114 patients and an antagonist protocol for 327 patients. The response to stimulation was monitored by serial hormonal blood tests and by ultrasound assessment of follicular and endometrial growth. Ovulation was triggered with human chorionic gonadotrophin (hCG) (Ovitrelle ® 250 μg MSD, Turkey) when at least two follicles had reached 17 mm. Oocyte pick up (OPU) procedures were performed 35 h after trigger hCG injection and cumulus cells were denuded 2 h after OPU. All oocytes were examined by inverted microscope with ×200 magnification just before ICSI. All assessments were done by the same embryologist and the findings in every single case were verified by the chief embryologist. Oocyte collection, ICSI and embryo transfer procedures and luteal phase support were performed as described elsewhere. 
Oocyte assessment and embryo grading
The patients were classified in two groups based on the appearance of the ooplasm: patients with all oocytes having centrally dark and granular cytoplasm were diagnosed to have degenerative signs (Group I) whereas patients with oocytes not having these features constituted Group II [Figure 1]. No other features of the oocytes regarding meiotic spindle, zona pellucida, polar body, oocyte shape and perivitelline space were taken into consideration in this study. The distribution of Group I and Group II oocytes in patients who were diagnosed with endometriosis and in those who had etiologies other than endometriosis were documented. The diagnosis of endometriosis was based on either laparoscopic confirmation or existence of endometriotiomas on ultrasound examination.
|Figure 1: Oocytes with centrally dark and granular cytoplasm (a) and with normal cytoplasm (b)|
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Embryos were graded on a scale of 1-4, 1 being the best, according to the criteria described by Balaban et al. 
Clinical pregnancy rates were compared between the groups.
Statistical analysis of data was performed by Statistical Product and Service Solutions (SPSS® IBM Corporation, NY, USA). Student's t-test and Chi-square test were used as appropriate. P value was considered as significant when <0.05.
The study was approved by the institutional review board and informed consent was routinely taken from all patients prior to enrolling the treatment program appropriately.
| Results|| |
Data of 792 patients were reviewed retrospectively. There were a total of 75 patients in Group I and 717 patients in Group II. Number of patients diagnosed with endometriosis was 62, while 730 patients had other infertility etiologies. Demographic and general characteristic features of the groups are depicted in [Table 1].
Number and the percentage of degenerative and normal oocytes related to endometriosis are given in [Table 2].
|Table 2: Number and the percentage of degenerative and normal oocytes related to endometriosis|
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The percentage of oocytes with degenerative signs is significantly higher in endometriosis (27.4%) group compared with control group (7.9%) (P < 0.001).
Although, there is tendency to lower pregnancy rate in endometriosis group (38.7%) the difference is not statistically significant (P = 0.077).
Pregnancy rates in endometriosis and non endometriosis cases and the relation of pregnancy rates with degenerative signs are displayed in [Table 3] and [Table 4] respectively.
| Discussion|| |
Commonly accepted features of a healthy oocyte based on morphological assessment are: single polar body, "normal-looking" cytoplasm, appropriate zona thickness and proper perivitelline space.  Embryos derived from normal or abnormal oocytes show different developmental capacities and the morphology of the oocyte may affect pregnancy outcome.  Since ovarian stimulation is a supraphysiological procedure, inherently compromised oocytes accompany the normal ones and they will have to be eliminated during the laboratory process.
Taking into consideration that up to 60-70% of the oocytes retrieved following controlled ovarian stimulation show abnormal morphological characteristics,  assessment of oocyte morphology gains utmost importance. Particularly severe abnormalities of the ooplasm have been shown to affect embryo quality more.  Hence, efforts have been spent to create a non-invasive, simple, cost effective and standardized selection method to select the proper oocytes in ART cycles. Recent techniques such as continuous time-lapse observation of embryo development, microarray and mass spectrophotometry are either extremely expensive or not easily applicable.
Predictive potential of morphological examination of human oocytes have been gaining importance as the data have been gathered more. Investigated structures of the oocyte are: meiotic spindle, zona pellucida, vacuoles or refractile bodies, polar body shape, oocyte shape, dark cytoplasm or diffuse granulation, perivitelline space, central cytoplasmic granulation. 
There is not a consistency in defining the abnormal features of the ooplasm. Various names are used including "dark cytoplasm," "dark cytoplasm-granular cytoplasm" "dark cytoplasm with slight granulation," "dark granular appearance of the cytoplasm" and "diffused cytoplasmic granularity." 
Some studies claimed that dark cytoplasm compromises the quality of the subsequent embryo , while many others did not find any adverse effect on in vitro or in vivo parameters. ,,, There is a lack of consensus about the influence of presence of granulation as well. Diffuse peripheral granulation was found to be associated with compromised pronuclear morphology after fertilization of these oocytes. , However interestingly, according to Wilding et al., any type of cytoplasmic granulation was associated with higher fertilization rates than in oocytes with total absence of granularity. 
Kahraman et al. investigated the impact of centrally located granular cytoplasm (CLGC) and did not find any correlation with fertilization rates, embryo development or pregnancy rates while ongoing pregnancy rates were seriously compromised.  In contrast, Wilding et al. have reported that oocytes with centrally located granulation were not found to have inferior fertilizing and in vitro developmental ability compared with those with completely absent granulation in the cytoplasm.  Balaban et al. in their study have reported that embryos derived from oocytes with vacuolar cytoplasm or central granulation do not seem to bear the potential to develop into good quality blastocysts or to reach hatching stage after cryopreservation.  Based on this study, it was claimed that extracytoplasmic structural changes should not be considered as abnormalities, but only phenotypic deviations from the normal, whereas severe cytoplasmic abnormalities should be considered to be genuine abnormalities resulting in poor developmental outcome of embryos derived from these types of oocytes.
In the present study, although the presence of dark and granular ooplasm does not compromise the embryo quality, it decreases the pregnancy rates significantly. Furthermore, we have found a relationship between the presence of the mentioned morphological abnormalities and endometriosis. The association of endometriosis with infertility is unequivocal; however, the mechanisms involved remain unclear. Some studies showed that even with no adhesions or anatomic distortions, endometriosis is associated with infertility; thus, apart from mechanical distortion of normal pelvic anatomy, immunologic defects and altered characteristics of the peritoneal environment, ovarian dysfunction is also an important contributing factor.  In this regard, our study caught another clue that endometriosis decreases fertility potential through compromising the oocyte quality. Although decreased embryo quality due to endometriosis is a well-known reality, to the best of our knowledge, ours is the first study defining a particular oocyte morphology related to endometriosis. Although there are several existing studies revealing correlation between some degenerative findings and endometriosis, our study emphasize that there is a strong relationship between particular cytoplasmic findings and endometriosis.
The appearance of a large, dark, spongy granulated area in the cytoplasm was defined as CLGC or as organelle clustering , and these central granulations together with vacuoles of the ooplasma may represent genetic, epigenetic or metabolic defects and shown to result in morphologically and/or genetically abnormal embryos. , It was claimed that intracytoplasmic organelle clustering is significantly repetitive in consecutive cycles. 
In their systematic review, Rienzi et al. summarized 50 studies which were published between 1996 and 2009 and focusing on consequences of morphological features of oocytes obtained with non-invasive methods. They did not find any tendency to a general increase in predictive value of morphological features. Of the 50 papers, 33 analyzed a single feature. Nine of the remaining 17 papers observing multiple features investigated the effect of these features individually, whereas eight papers summarized the effect of individual features. Interestingly, none of the investigated nine single features was unanimously correlated with normal or compromised development, when evaluated by 15 outcome parameters. Among all abnormal features of oocyte, central granulation seem most consistently to be correlated with compromising effect on further development. 
Several mechanisms are postulated in order to explain the adverse effect of the abnormal ooplasma on clinical outcome parameters such as increased rate of aneuploidy found in the embryos derived from such oocytes , and lower intracytoplasmic pH and adenosine triphosphate content. 
Morphological variations of the oocyte may result from intrinsic factors such as age and genetic defects or extrinsic factors such as stimulation protocols, culture conditions, and nutrition. In the present study, in addition to contribute to the existing data regarding the adverse effect of the dark and granulated ooplasm on a certain outcome variables of ICSI cycles, we reveal the relation of the mentioned oocyte features with endometriosis. This finding strengthens the theory about the causal effect of endometriosis on poor oocyte quality.
| Conclusion|| |
The incidence of encountering oocytes with dark and granulated ooplasm is higher in patients with the diagnosis of endometriosis compared with the infertile couples diagnosed with non-endometriosis etiology. This finding can be used as a clue in cases in which the presumptive diagnosis of endometriosis has not been verified by laparoscopy. The typical morphology also has an adverse effect on clinical pregnancy rate.
The abstract of this study was presented in the 28 th annual ESHRE meeting held in June 2012 in Istanbul.
| Acknowledgment|| |
Many thanks to Can Deniz Kφksal from Akdeniz University for valuable comments and statistical revision of the manuscript.
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| Authors|| |
Ayse Kendirci Ceviren graduated from Cukurova University Faculty of Biology in 1985 and soon after started studying in the field of reproductive medicine and infertility. She took part in the foundation of Cukurova University IVF Center as well as some private IVF centers in Turkey and in Northern Cyprus Turkish Republic. She trained ICSI and embryo vitrification in Israel. Her current position is the chief embryologist in Gelecek The Center For Human Reproduction.
[Table 1], [Table 2], [Table 3], [Table 4]
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